Protein mixtures can be difficult to resolve using only one separation technique. Therefore, two-dimensional or multidimensional separations are sometimes used. Two-dimensional refers to the fact that the sample mixture is partially resolved (in one dimension) using one separation technique, then the output from this first separation is further resolved (in the second dimension) using a second separation technique. The number of dimensions is equal to the number of separation techniques employed. The sample properties that determine sample separation in the first dimension should be different from those properties that determine sample separation in the second dimension in order to maximize separation resolution. If the sample properties that determine separation are totally different in both dimensions, the dimensions are said to be orthogonal. This is desirable since it enhances separation resolution.
An example of a two-dimensional separation is described by Liu and Le Van in U.S. Patent Application Publication U.S. 2002/0033336 A1. The first dimension is high-performance liquid chromatography (HPLC) and the second dimension is a plurality of electrophoresis columns. Liu and Le Van also disclose a separation where the first dimension is isoelectrical focusing and the second dimension is an array of capillary gel electrophoresis channels.
Another example of a two-dimensional separation is described by Wiktorowicz and Raysberg in U.S. Pat. No. 6,013,165. In one embodiment of the invention, the first dimension is gel electrophoresis to separate samples by size and charge and the second dimension is isoelectric focusing.
Akins in U.S. Patent Application Publication No. US 2002/0153252 A1 describes further examples of 2-dimensional systems in which the first dimension is cationic electrophoresis and the second dimension is one of denaturing electrophoresis, electrophoresis subsequent to proteolytic cleavage, isoelectric focusing non-equilibrium pH gel electrophoresis or immobilized pH gradient electrophoresis.
The present invention is an orthogonal two-dimensional system employing chromatofocusing (CF) as the first dimension and multiplexed capillary gel electrophoresis (MCGE) as the second dimension. These two dimensions are totally orthogonal, unlike some of the others above mentioned and, therefore, result in a higher degree of separation resolution.
For reasons not fully known to the inventors, no one has previously combined CF and MCGE as the two dimensions. Perhaps this is because they are relatively new techniques, their orthogonal nature has not been appreciated, and some of the buffer reagents used for each have been incompatible. Applicants have, however, discovered that the combination of CF and MCGE achieves good resolution in minimum time and can be used to advantage.
The widely accepted technique for protein analysis is traditional 2D gel electrophoresis. This is a method for the separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right angles to one another. The first dimension is isoelectric focusing (IEF) which separates proteins according to isoelectric point (pI) differences while the second dimension is polyacrylamide gel electrophoresis (SDS-PAGE) which separates proteins according to their sizes.
However, there are many disadvantages related to the 2D gel electrophoresis. It is labor intensive, time consuming and poorly automated. Usually it takes several days to complete an analysis. Proteomics research requires the development of new techniques that have the following features: (1) increased resolving power and speed, (2) the ability to analyze proteins with varied properties (isoelectric points, molecular weights, hydrophobicities), (3) simplicity and automation and (4) the ability to perform high throughput analysis.
CF coupled with MCGE is a good alternative for the traditional 2D gel electrophoresis. It provides higher speed (it takes several hours to complete an analysis instead of several days in traditional 2D gel electrophoresis), automation and high throughput. The data output is directly comparable to the traditional 2D gel electrophoresis results.
CF is a form of ion-exchange chromatography. The objective of CF is to elute proteins from a column in order of their isoelectric points. An isoelectric point is the pH at which the net charge on a molecule in solution is zero. A weak anion (in anion CF) exchange column is equilibrated with a low ionic strength buffer at a high pH. The sample protein is loaded onto the column. Proteins are bound to the anion exchanger at the high pH. A pH gradient is then produced by adding a second, lower pH buffer. This buffer contains species that have a wide range of pKas. The range of pKas provides level buffer capacity across the entire pH range of the gradient. As the pH on the column decreases, protein positive charges become stronger and there is less interaction between the column and the protein. Eventually, the protein does not interact with the column and it elutes. The bound proteins are eluted in order of their isoelectric points, from high to low.
High performance MCGE has rapidly become an important analytical tool for the separation of a large variety of compounds ranging from small ions to large biological molecules. MCGE is used for general separations, enantiomeric separations, protein separations, the peptide mapping of proteins, amino acid analysis, nucleic acid fractionation and the quantitative measurement of acid dissociation constants (pKa values) and octanol-water partition coefficients (log Pow values).
What all these MCGE applications have in common is the measurement of the mobility of chemical species in a capillary tube as a means of identifying it. To perform a conventional separation, a capillary tube is filled with a buffer solution, a sample is loaded into one end of the capillary tube, both ends of the capillary tube are immersed in the buffer solution and a large potential is applied across the capillary tube. The sample components are separated electrophoretically as they migrate through the capillary tube. In a UV detection system, a section of capillary tube is irradiated with a UV light source. A photodetector detects the light that passes through the tube. When a UV absorbing sample component passes through the irradiated portion of the capillary tube, the photodetector detects less passed light (indicating absorbance). In this way an electropherogram, a plot of absorbance versus time, can be produced.
The rapid development of biological and pharmaceutical technology has posed a challenge for high-throughput analytical methods. For example, current development of combinatorial chemistry has made it possible to synthesize hundreds or even thousands of compounds per day in one batch. Characterization and analysis of such huge numbers of compounds has created a bottleneck. Parallel processing (i.e., simultaneous multi-sample analysis) is a natural way to increase the throughput. Unlike high-performance liquid chromatography or gas chromatography, it is practical to build a highly multiplexed CE instrument that can analyze dozens of samples simultaneously. Such a system has been disclosed in PCT Application WO 01/18528A1.
There is a continuing need for development of multidimensional separation techniques of high speed and high resolution. To date, no one has combined chromatofocusing (CF) and multiplexed capillary electrophoresis (MCGE). It is believed that this is because both techniques are relatively new; chromatofocusing was disclosed in 1978 and MCGE is even younger and because their orthogonal relationship has not heretofore been appreciated for use in two-dimensional techniques.
Another reason that CF and MCGE have not been combined for protein separation is that buffers used for CF often interfere with the absorption detection employed with MCGE. Protein absorbance is stronger at a wavelength of 214 nm than 280 nm. Therefore, 214 nm is preferred for MCGE detection systems because it allows greater sensitivity of detection. However, typical CF systems use absorbance detection for proteins at 280 nm. The reason is that the buffer used, commonly Polybuffer™ available from Amersham BioSciences, strongly absorbs at 214 nm. If Polybuffer™ is used in conjunction with a detection system at 214 nm, the absorbance distorts the baseline and hinders detection of proteins. In short, one reason the two techniques have not been combined is a lack of a buffer that will work well in both systems, preferably at 214 nm. The applicants have discovered such a buffer.
The applicants have discovered that multidimensional separations combining CF and MCGE as herein described have the advantage of being totally automatable, thus achieving certain labor efficiencies. Furthermore, it is advantageous to combine CF rather than isoelectric focusing, as has been done in the past, with MCGE. This is because CF has the capacity to handle large samples. This is beneficial to the second dimension, MCGE, for detection and separation. If the amount of sample from the first dimension is too low, there can be sensitivity problems in the second dimension.
Additionally, it is particularly advantageous to combine CF with MCGE because the output from CF is a large number of aliquots of solution. With MCGE, due to the multiplexing, all the aliquots can be analyzed simultaneously in separate capillary tubes.
The primary objective of the present invention is to design a two-dimensional, orthogonal separation technique that combines CF and MCGE to provide high speed and high resolution separations. The method and manner of achieving this primary objective as well as others will become apparent from the detailed description that follows.